Blebbistatin was discovered in a screen for chemical inhibitors of nonmuscle myosin IIA (NMIIA) ATPase activity. It inhibits blebbing and cytokinesis in Xenopus and human cells. We tested its activity against a number of other conventional and unconventional myosin isoforms. Blebbistatin inhibited the actin-activated MgATPase activity of NMIIA heavy meromyosin (HMM), NMIIB (HMM), smooth muscle HMM, and several striated muscsle muscle HMMs. The amount of blebbistatin required for half maximal inhibition of the actin-activated MgATPase is ranged form 0.4 micromolar for rabbit skeletal muscle HMM to 80 micromolar for smooth muscle HMM. Interestingly NMIIA and NMIIB were both half maximally inhibited by 3-5 micromolar blebbistatin even though these molecules are closely related to smooth muscle myosin. Blebbistatin, even at 100 micromolar, did not effectively inhibit the activity of rat myo1b, Acanthamoeba myosin IC, myosin-V and myosin-X. Blebbistatin completely inhibited the movement of rhodamine-phalloidin labeled F-actin by NMIIA and skeletal muscle HMM. This effect was reversible upon wash out of blebbistatin. The inhibitory activity of blebbistatin was destroyed by illumination with blue light (488 nm). This property could be useful in studying the reversibility of blebbistatin inhibition of contractile processes in cells and for synchronizing contractile events. We have done a preliminary characterization of the kinetic mechanism for the blebbistatin inhibition of myosin. The inhibition is complex and probably involves blebbistatin binding to a myosin nucleotide complex which subsequently inhibits the binding of a new ATP after the dissociation of the originally bound nucleotide.